Genetic divergence in two NODk mice strains with varied diabetes penetrance

Abstract

Diabetes mellitus is a chronic metabolic disorder characterized by persistent hyperglycaemia due to inadequate insulin secretion (beta-cell dysfunction) and/or impaired insulin action (insulin resistance). While T1D is caused by autoimmune destruction of islet beta-cells, islet beta-cell dysfunction often with progressive decline in beta-cell mass contributes to pathogenesis of T2D. Recent evidence of clinical convergence of T1D and T2D phenotypes in a proportion of people with diabetes suggests possible shared non-immune islet susceptibility factors. The T1D-resistant O-NODk mouse sub-strain (created from the T1D-prone NOD mice through replacement of the Idd1-H2g7 with Idd-H2k) develops impaired glucose tolerance, hyperinsulinaemia and T2D when fed a high fat (HF) diet. Through routine colony refreshment, our laboratory developed another sub-strain of NODk mice, the RF-NODk mice, which are resistant to HF diet-induced T2D. Since both O-NODk and RF-NODk mice colonies were maintained in the same animal facility under similar environmental conditions, we hypothesised that T2D predisposition in the O-NODk mice is of genetic origin. Thus, the aim of this project was to characterize the phenotype and genotype of both NODk mice sub-strains to determine the genetic factor(s) causing T2D susceptibility in O-NODk mice. A long-term study (from 4-24 weeks of age) in these two mice sub-strains fed normal chow (NC) or HF diet monitored body weight, blood glucose and plasma insulin fortnightly and pancreatic histological alterations were evaluated at the end of the study. Assessments of beta-cell function were performed in vivo and in vitro through intraperitoneal glucose tolerance tests (ipGTT) and isolated islet static insulin secretion, respectively. A cross-breeding study was designed to determine the allelic frequency and segregation of the diabetes susceptibility genetic factor(s) in the O-NODk mice. Whole genome sequencing (WGS) and RNA sequencing (RNA-seq) were performed to identify genetic variants in the exome and transcriptomic profiles of these two mice sub-strains, respectively. HF-fed O-NODk mice developed accelerated weight gain, impaired glucose tolerance, severe hyperinsulinaemia and T2D, whereas RF-NODk mice maintained normoglycaemia. Pancreas histological analysis revealed increased exocrine pancreas fat infiltration, mildly increased peri-ductal inflammation, and a similar mild peri-islet inflammation compared to RF-NODk mice. Reduced beta-cell mass was observed in diabetic HF-fed O-NODk mice. In vitro studies revealed that pancreatic islets of HF-fed O-NODk mice secreted more insulin in response to glucose and fatty acids, and the insulin secretagogue IBMX. Cross-breeding studies showed that the diabetes trait in the O-NODk mice segregated as a simple Mendelian homozygous recessive trait. WGS identified 6 candidate genes with single nucleotide polymorphisms (Atp13a3, Kcnh7, Gpr125, Ncor1, Myh4 and Olfr954) and over 1200 insertions and deletions in O-NODk mice only. RNA-seq highlighted the up-regulation of Sdc2, Suclg2 and Cbr3 genes, and down-regulation of Pcp4 in O-NODk mice. In conclusion, the phenotype of the diabetes-prone HF-fed O-NODk compared to diabetes resistant HF-fed RF-NODk mice, includes accelerated weight gain, severe hyperinsulinaemia, enhanced in-vitro insulin secretion, dysfunctional in-vivo insulin secretion and late loss of beta-cell mass. Genetic studies suggest Mendelian homozygous recessive inheritance. Several candidate genes have been elucidated which warrant further validation and mechanistic studies. Show more